The invention relates to methods and devices for applying substances to a support, especially monomers for the combinatorial synthesis of molecule libraries as used in the detection of optical properties, more especially of luminescence reactions and refraction behaviour, of molecules bound on the support.
In the following description, the term “molecule library” denotes the entirety of many different molecules bound at defined places on a support, whereby the various molecules are arranged as compactly as possible. The molecule libraries to which the invention relates are formed hereby more especially by the combinatorial synthesis of a limited number of monomers. The principle of combinatorial synthesis is explained schematically in FIG. 1.
The term “highly complex” denotes molecule libraries having more than 103 different representatives, more especially however molecule libraries having more than 105 different representatives.
Said complex molecule libraries can be applied particularly advantageously to a two-dimensional support whereby each different member of the molecule library can then be allocated a locally precisely defined place on the support. This means that a locally precisely defined reaction e.g. a staining reaction permits exact and unique conclusions to be reached on the support-bound reaction partner. The locally precisely defined places with the defined member of said molecule library are also called spots and the entirety of the molecule libraries on the two-dimensional support are also called arrays.
The two-dimensional array can have a smooth surface and be essentially impenetrable for the solvents used. However, it can also exhibit a porous structure compared thereto so that a third dimension is revealed to the solvents used and the substances to be linked. Supports of this type are particularly indispensable if the signal strength is to be increased compared with a support having a smooth surface. This is particularly the case when, for example, an array of peptides is to be stained with the blood serum of a patient and the binding signals of relatively weakly concentrated antibody reactivities are also to be detected.
Subsequently the synonymous terms “two-dimensional support” or “array” thus mean both supports where different molecules are essentially arranged in only two dimensions and also porous supports where the various molecules are present in an additional third dimension, thus are no longer (essentially two-dimensional) arrays in the real sense.
The term “solid state of aggregation” also includes undercooled liquids.
The term “properties” is understood in the broadest sense and should include not only the properties characteristic of specific molecules, such as for example their mass spectrogram but, for example, also the capability in general, namely by the mere presence, of displaying a certain reaction so that the invention thus also relates to such methods and devices for which initially only the mere presence of a substance, but not its type, should be concluded from a particular optical reaction (whereby the type of substance is then determined for example from its position on the support).
The term “biological” molecules is here taken to mean all types of molecules particularly relevant in biology, pharmacy and medicine, thus for example, peptides, D-peptides, L-peptides and mixtures thereof, naturally occurring oligonucleotides, their mirror images and mixtures thereof, artificially derivatised oligonucleotides, such as those used for construction of aptamers, oligosaccharides and modifications of said molecules. More especially, modular constructed oligomers which do not occur in nature can have particular pharmacological relevance. Particular mention in this connection may be made of non-natural substances produced with the aid of chemical combinatorial analysis which can be used as ligands of biological molecules, more especially organic compounds, steroid derivatives and so on. From many of these molecules specific binders can be isolated for a naturally occurring molecule which modify the activity of this molecule. However, since these binders frequently cannot be detached from naturally occurring digestive enzymes, they are especially suitable for use as therapeutics.
Various methods and devices are known for the synthesis of highly complex molecule libraries but these possess certain disadvantages. Thus the known methods require costly and expensive special equipment for their implementation and are comparatively slow in the readout of a luminescence signal. In particular, if, as is advantageous for different reasons as will be explained subsequently, very many different molecular groups are to be arranged on a common support and investigated singly, very expensive mechanics must be used to activate the individual molecular groups, which is not only expensive and liable to breakdown but also always exhibits manufacturing tolerances in maximum precision work, which are several orders of magnitude higher than the minimum size of the molecular groups sufficient for an investigation. As a result, the maximum number of molecules or molecular groups to be accommodated on a support is limited for known methods and devices and is roughly in the order of magnitude of a few 105 molecular groups. In particular for certain blood serum or DNA analyses, however, it would be desirable if approximately 108 to 109 molecules could be accommodated and studied on one support.
FIG. 2 shows the principle of the confocal laser microscope which is used for readout in the current, especially lithographic methods of synthesis. It can be seen that in this readout mechanism every single point of the array must be searched in all three dimensions which either costs time or accuracy.
Lithographic methods are known for applying molecules to the appropriate supports, especially to so-called “diagnostic chips” (FIG. 3) whereby however, as in the later investigation, the difficulty of exactly assigning molecules and reproducibly purposefully activatable support positions limits the maximum number of molecules which can be applied, since it is not sufficient to arrange very many different molecules closely packed on a support without knowing, however, with reproducible accuracy which molecules are located in which position on the support. In particular, in the known methods and devices reading out very many luminescence reactions on a support in a reasonable time and at the same time very accurately is a problem. In the staining investigations which can be carried out advantageously using the methods and devices with which we are concerned here, in which a material to be examined is applied to a support on which different molecules have already been anchored, conclusions should be reached on the substances present in the material to be examined, such as for example specific antibodies in a blood serum, with which the molecules of the material or its constituents anchored on the support have formed bonds so that one must know very accurately which molecule is located where on the support.
In addition, all known lithographic methods (and some other methods where, for example, locally precise synthesis is achieved by the controllable repulsion or attraction of electrically charged monomers) have another fundamental disadvantage: for each of the different monomers almost the entire linking cycle must be run through separately, i.e. each type of monomer is applied, linked and excess monomers washed away, followed by the next type of monomer so that, for example, in the combinatorial peptide synthesis layer for layer 20 linking cycles must be run through in each case. This disadvantage is shown schematically in FIG. 4. Thus, for the synthesis of a complex pentapeptide library these methods require 100 linking cycles whereby the expert can immediately appreciate that at the present state of technological development this will lead to serious quality problems for the resulting molecule libraries as a result of the artefacts to be expected in each linking cycle so that pentapeptide libraries produced in this way are in fact unusable.
This is also the reason why the lithographic methods have so far been used almost exclusively for the synthesis of oligonucleotide arrays since in this case only four different monomers need to be linked to the support.
Another side effect is the comparatively poor yield of chemicals in the lithographic methods since for each linking reaction the entire support must be covered uniformly with the reactive monomers.
In addition to the lithographic methods, there are also many printing methods which can be used to carry out combinatorial syntheses (FIG. 5, IIa & IIb). So far however, none of these methods reaches the high resolution of the lithographic methods. The reason for this is mainly the high rate of diffusion of the relatively small monomers in solution. Since a certain time is always required both for the linking reaction of the monomers to the support and for the application of the monomers to the support in precise positions, the high diffusion rate thus limits the attainable compactness of the molecule libraries produced by combinatorial synthesis and therefore also their complexity.
A comparison with a normal color ink jet printer should clarify this argument (FIG. 6): the brilliance of the color imprints of color ink jet printers is achieved by keeping the diffusion of the various color particles as low as possible. This is achieved by the enormous size of the color particles compared with the afore-mentioned monomers and by the printed toner fluid containing rapidly volatile substances so that the color particles are precipitated very quickly. In addition, special highly absorbent high-gloss paper is used.
These papers generally having a complex structure are not usually suitable as supports for a molecule library and also the two other points are not consistent with the requirements for linking a molecule library as closely packed as possible to the support:
1. The monomers for combinatorial synthesis are very much smaller than the normally used color chromophores of a color ink jet printer and this fact alone increases the diffusion rate enormously.
2. Not only can the printed monomers not be dissolved in highly volatile solvents. It is barely even feasible to find a solvent that does not vaporise too rapidly in the desired quantities in the nanoliter range since the concentrations of the linking partners would thus change in an undesirable fashion because the linking reaction to the support (and the application of the monomers to the support in precise positions) requires a certain time.
This is the reason why all the spot methods used so far are liable to error and expensive as soon as they are used in smaller dimensions. In these dimensions there is always the risk that the applied spots run, the monomers diffuse too far or the solvent volatilises partly or completely.
It would therefore be desirable and advantageous to provide a method and a device to obviate prior art shortcomings in the synthesis of molecule libraries on supports.